Drug discovery efforts generally focus on the identification of compounds that modulate, inhibit or enhance the activity of the target of interest. Conventional lead identification efforts proceed via biochemical or cell based screening or in silico compound design. These methods have identified and validated a multitude of viable therapeutics in use today. However, as reflected by the high failure rate of new drug compounds (only an estimated 8% of phase I clinical therapeutics eventually gain Food and Drug Administration approval, at a conservative cost of $800 million per drug), many efforts are unsuccessful and often targets are abandoned once they are deemed undruggable (Lee et al., 2009). A considerable part of these failures are due to the fact that most biochemical or cell based assays are performed on targets in their prominent conformation, also referred to as the basal conformation. However, we now know that conformational flexibility is key to the function and the pharmacology of the majority of the current and future drug targets including GPCRs, ion channels, (nuclear) receptors, kinases and phosphatases. And for many of these targets, the most stable conformation, corresponding to the prominent structural species in the absence of ligands or accessory proteins (the basal conformation), does not correspond to the druggable conformation to which a drug must bind to be most effective for the therapeutic indication.
Today, the most commonly targeted protein class for medicinal intervention are G protein-coupled receptors (GPCRs), also called seven-transmembrane receptors (7TMRs). They play essential roles in physiological responses to a diverse set of ligands such as biogenic amines, amino acids, peptides, proteins, prostanoids, phospholipids, fatty acids, nucleosides, nucleotides, Ca2+ ions, odorants, bitter and sweet tastants, pheromones and protons (Heilker et al., 2009). Orthosteric ligands that act on a GPCR can induce a spectrum of effects on down-stream signaling pathways. In general, GPCRs require agonist binding for activation. Full agonists maximally activate the receptor. Partial agonists elicit a submaximal stimulation even at saturating concentrations. In some cases, a GPCR may exhibit basal activity towards a specific signaling pathway even in the absence of an agonist (constitutive activity). Inverse agonists can inhibit this basal activity. Notably, whereas neutral antagonists can inhibit binding of agonists, partial agonists, and inverse agonists at the orthosteric binding site of GPCRs, they do not alter the basal receptor activity. In recent years, advances have been made in the discovery of novel ligands for GPCRs that act at allosteric sites to regulate receptor function, including positive and negative allosteric modulators (PAMs and NAMs, respectively) and neutral ligands, which offer novel modes of action over orthosteric ligands (Christopoulos 2002).
It is now well established that GPCRs can signal through several distinct mechanisms including those mediated by G proteins or the multifunctional adaptor proteins β-arrestins (Rajagopal et al., 2010). With the structures of several GPCRs solved in complex with various ligands including inverse agonists, antagonists and agonists (Cherezov et al., 2007, Rasmussen et al., 2011b, Rosenbaum et al., 2011, Shimamura et al., 2011, Xu 2011, Granier et al., 2012, Haga et al., 2012, Hanson et al., 2012, Kruse et al., 2012, Manglik et al., 2012, Wu et al., 2012, Zhang et al., 2012) and the G-protein (Rasmussen et al., 2011a), we now know that GPCRs are conformationally complex molecules with specific conformations causing G protein activation. Of special significance in the context of this disclosure is the observation that in comparison to the basal conformation, only relatively small changes in the structure of the agonist binding pocket led to substantial movement (up to 14Å) and rearrangements in three of the transmembrane segments (Lebon et al., 2012).
Mass-spectrometry-based strategies (Kahsai et al., 2011), biophysical analysis (Yao et al., 2006, Mary et al., 2012) and NMR spectroscopy (Liu et al., 2012; Bokoch et al., 2010) provide direct evidence for the presence of other distinct ligand-specific conformations that lead to arrestin mediated signaling. It follows that different ligands can have differential effects on the conformation and the diverse signaling and regulatory repertoire of a single receptor. The importance of these multiple conformational states is their pharmacological relevance. As illustrated in FIG. 1, each of these receptor conformations can be considered as a separate therapeutic drug target because each of these conformations promotes distinct relative efficacies toward the different effector systems including G proteins and arrestins.
Drug discovery approaches can take considerable advantage from capturing the target in a therapeutically relevant “druggable” conformation. Stabilizing a receptor in a particular functional conformation would inherently freeze the receptor in a single, disease relevant druggable conformation revealing new structural features that are suitable for targeting with small molecules or biologicals and may enable the identification of compounds that are selective for that druggable conformation. The stabilization of a unique druggable conformation, including inactive states corresponding to effector systems below basal activity or particular functional states that activate individual effector systems could not only lead to compounds with better therapeutic efficacies but could also benefit the identification of compounds with less undesirable side effects that result from triggering undesired pathways (Galandrin et al., 2007).
Further to that, conformational flexibility is an issue in high-throughput screening (HTS) and fragment-based drug discovery (FBDD) (Lawson 2012). In HTS, issues of different conformations of a target can in some cases be overcome by using whole-system assays with a functional readout rather than reductionist recombinant systems assays (Kenakin, 2009; Rajagopal et al., 2010). In FBDD, however, whole-system assays cannot be used because of the low efficacy/potency of the initial hits, often requiring mM concentrations of the fragments for biological activity and causing toxicity. It follows that high-throughput primary screens would benefit considerably from target receptors that are stabilized in the desired functional conformation in the absence of ligands or accessory proteins. Access to such conformationally stabilized receptors would allow the identification of the subset of ligands that are specific for that conformation with its particular structural features (FIG. 1). In this way, a first selection of the potentially biologically active compounds can be made using simple assessment of binding before establishing their efficacy profiles in a variety of (whole-system) signaling assays.
Conformational flexibility also obstructs structure based drug discovery starting from fragments. First, many of the potential hits of fragment-based screening (FBS) are not potent enough to quantitatively displace the conformational equilibrium into a single conformation of the protein-ligand complex that can be crystallized in a diffracting crystal. If a complex cannot be crystallized, soaking existing crystals of ligand-free protein with (small) ligands is often the method of choice to obtain crystals of the complex. However, if these ligands displace the conformational equilibrium of a conformationally complex protein, these induced conformational changes will in many cases destroy the crystals (Danley 2006).
With the structures of the first GPCRs solved in 2007 (Rasmussen et al., 2007, Rosenbaum et al., 2007), we entered the new era of GPCR structural biology raising the possibility of applying structure-based approaches to GPCR drug discovery efforts (Shoichet and Kobilka 2012). For a large number of GPCR drug targets relating to several therapeutic indications, the agonist-bound active-state is often the druggable conformation. Resolving the structure at high resolution of a GPCR in this therapeutically relevant “druggable” conformation remains a challenge. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. Structures of GPCRs in complex with full agonists have been solved but not without difficulties. First, natural agonists generally do not sufficiently stabilize the receptor for the formation of diffraction-quality crystals. In an attempt to solve this problem, agonists have been covalently bound to GPCRs for crystallization purposes. However, for example, the crystal structure of a covalent agonist-bound β2AR reveals a conformation closely resembling an inactive state rather than the active state with only little rearrangements in the transmembrane segments (Rosenbaum et al., 2011) (Lebon et al., 2012).
Another approach towards determining agonist-bound conformations of a GPCR is thermostabilization of the receptor via systematic mutagenesis followed by measuring increased thermostability in the presence of bound agonist (e.g., WO2008114020, WO2009071914, WO2010149964, WO2012098413). For example, thermostabilizing mutations have been discovered for the agonist-bound A2AAR (Lebon et al., 2011), the agonist bound β1-adrenergic receptor (Warne et al., 2011) and the agonist bound neurotensin receptor (White et al., 2012). However, the structures of these agonist bound stabilized receptors are likely not in the fully active conformation, judged on the small displacement of transmembrane helix 6. More important and in contrast to the active state that is stabilized by a G-protein or a G-protein mimic, these thermostabilized receptors show no significant increase in the affinities for their respective agonists (Serrano-Vega et al., 2008, Shibata et al., 2009, Lebon et al., 2011).
Only recently, it became possible to obtain structures of an agonist-bound active state of a GPCR, making use of conformationally selective Nanobodies (XAPERONE™) that mimic G protein function and increase the affinity for agonists at the orthosteric site (Rasmussen et al., 2011b). XAPERONES™ are useful tools to lock the structure of GPCRs in a therapeutically relevant conformation (Steyaert & Kobilka, 2011) and facilitate the discovery of drug candidates by increasing the sensitivity and selectivity of existing screening methods (WO2012007593). However, this technological approach also has its limitations. Because the binding of the agonist at the orthosteric site increases the affinity for the G-protein mimicking XAPERONE™ at the allosteric intracellular side of the receptor and vice versa (Rasmussen et al., 2011b), the GPCR-XAPERONE™ complex is much more stable in the presence of an agonist. It follows that a GPCR, a stabilizing XAPERONE™ and agonist have to be co-crystallized to obtain crystals of the GPCR in its active conformation and that the disclosure described in WO2012007593 is not very well suited for structure based drug discovery approaches involving, for example, soaking of ligands in existing crystals because the agonist that has been used to grow the crystals will compete with the ligand that is soaked in subsequently.
Thus, the development of new methods that constitutively stabilize GPCRs in a particular druggable conformation, even in the absence of agonists, would be an important asset to improve drug discovery via compound screening and/or structure based drug design.